High-performance liquid chromatography (HPLC) analysis of hemoglobins is a widely used technique. Specifically, this technique is used, for example, to quantify a glycohemoglobin known as hemoglobin A1c or to analyze abnormal hemoglobins for diagnosis of diabetes. For example, a method utilizing liquid chromatography has been known which separates hemoglobin components in a diluted hemolyzed blood sample by a cation-exchange method based on the difference in positive charge among the hemoglobin components. A recent increase in patients with diabetes has also increased the number of cases requiring measurement of hemoglobin A1c. This tendency has created a demand for more accurate, less time-consuming measurement by HPLC.
Hemoglobins are present in the body in the forms of oxyhemoglobin that contains bound oxygen, deoxyhemoglobin that contains bound carbon dioxide, and methemoglobin in which the iron in the heme group is oxidized into the trivalent ion state. In the case of cation-exchange HPLC, oxyhemoglobin, deoxyhemoglobin, and methemoglobin may differ from one another in retention in a separation column and therefore may differ from one another in elution time as well. Consequently, the analysis may provide poor separation accuracy (e.g. detection of broad elution peaks or elution peaks in a bimodal distribution). Moreover, it is known that in the presence of an azide or cyanide, methemoglobin is converted to stable azide methemoglobin or cyanomethemoglobin (hereinafter, also referred to as stable methemoglobin) as a result of binding of the azide or cyanide to the trivalent iron ion in methemoglobin. Since the retention in a separation column is also slightly different among the stable methemoglobin, oxyhemoglobin, and deoxyhemoglobin forms, the presence of stable methemoglobin may cause a broad elution peak or an elution peak in a bimodal distribution to be detected.
HPLC analysis of hemoglobins is mainly used for diagnosis of hemoglobinopathy and thalassemia which may cause anemia, in addition to diabetes. Especially, the number of cases requiring separation and detection of hemoglobin S is large because hemoglobin S is the most common abnormal hemoglobin and causes sickle cell disease which results in severe anemia. On the other hand, in the case of measurement of the diabetes marker hemoglobin A1c, it is preferred to separate abnormal hemoglobins including hemoglobin S in sharp elution peaks. If hemoglobins are eluted in broad elution peaks or elution peaks in a bimodal distribution, separation of the abnormal hemoglobins from normal hemoglobins is difficult and this difficulty may cause a negative impact on obtained measurements.
Furthermore, deteriorated blood samples tend to give broad elution peaks or elution peaks in a bimodal distribution compared to fresh blood samples. This is because the amount of methemoglobin is increased due to deterioration. Therefore, in the case of analysis of a preserved sample (e.g. re-examination), there is a possibility of a negative impact on obtained measurements.
Addition of known antimicrobial agents or saccharides has been known as a technique to prevent hemoglobins from deteriorating and denaturing. Other ways to achieve it are, for example, coexistence of hemoglobins with albumin (Patent Literature 1); coexistence of hemoglobins with casein, boric acid, or the like (Patent Literature 2); and coexistence of hemoglobins with iminocarboxylic acid or a salt thereof (Patent Literature 3).
However, no matter which technique is used among the techniques of Patent Literatures 1 to 3, HPLC measurement of a blood sample containing hemoglobins in various forms including oxyhemoglobin, deoxyhemoglobin, and methemoglobin possibly results in broad elution peaks or elution peaks in a bimodal distribution.
Common HPLC instruments are equipped with a degasser for removing gas in eluents. Such a degasser may cause fluctuations in elution times of hemoglobins and have a negative impact on separation of hemoglobins because its gas removing performance is not stable for a while immediately after start-up. Especially, immediately after start-up or when the temperature of an eluent is unstable, the HPLC instruments are likely to cause fluctuations in elution times of hemoglobins and deterioration of quantification accuracy. This extends the time before a first report of analysis for diabetes diagnosis using hemoglobin A1c as a marker although such analysis requires a rapid result. Therefore, there is a need for a technique for providing stable measurement even immediately after start-up of an HPLC instrument.
Generally, absorbance obtained by a spectrophotometei is a measure to detect and quantify hemoglobins. The coexistence of oxyhemoglobin, deoxyhemoglobin, methemoglobin, azidemethemoglobin, and cyanmethemoglobin may inhibit accurate quantification of hemoglobins because of their different absorption spectra.